title: Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors.
abstract: The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr¹⁸⁴ in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr¹⁸⁴ depends on local residues, we generated mutations in an α7/5HT(3A) (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured ¹²⁵I-labelled α-btx binding. The results show that mutations of individual residues near Tyr¹⁸⁴ do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr¹⁸⁴ to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr¹⁸⁴ to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr¹⁸⁴ and local residues contributes to high-affinity subtype-selective α-btx binding.
authors.affiliation: Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55902, USA. sine@mayo.edu
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sine
authors.forename: Sun
authors.initials: S
authors.lastname: Huang
authors.forename: Shu-Xing
authors.initials: SX
authors.lastname: Li
authors.forename: Corrie J B
authors.initials: CJ
authors.lastname: daCosta
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000737:chemistry / Q000378:metabolism*; D017838:Bungarus; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D030162:Reptilian Proteins / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry*; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D002038:Bungarotoxins; C579167:Chrna7 protein, human; C509409:HTR3A protein, human; D008024:Ligands; D050505:Mutant Proteins; D009498:Neurotoxins; D010446:Peptide Fragments; D020033:Protein Isoforms; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D030162:Reptilian Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; D014443:Tyrosine